Biosynthesis and Insertion of the Molybdenum Cofactor

AXEL MAGALON^1* AND RALF R. MENDEL²

[SECTION EDITOR: T. BEGLEY]

Posted January 18, 2008

Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et de Microbiologie, CNRS, 13402 Marseilles Cedex 20, France,¹ and Department of Plant Biology, Technical University, 38106 Braunschweig, Germany²

*Corresponding author. Mailing address: Laboratoire de Chimie Bactérienne, Institut de Biologie Structural et Microbiologique, CNRS, 13402 Marseilles Cedex 20, France. Phone: +33-491-164-668, Fax: +33-491-718-914, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

INTRODUCTION

The transition element molybdenum (Mo) is of essential importance for biological systems because it is required by enzymes catalyzing key reactions in the global carbon, sulfur, and nitrogen metabolism (4, 49). But tungsten is also biologically important. Both elements have a very rich redox chemistry, which might explain why they are the only members of the second (molybdenum) and third (tungsten) transition series with known biological functions. Molybdenum is very abundant in the oceans in the form of the MoO₄²⁻ anion, while the tungsten concentration (as tungstate) is 100-fold lower. Under anaerobic conditions and high sulfur concentrations that prevail in deep-sea hydrothermal vents, molybdenum occurs as MoS₂ and thus becomes unavailable for biological systems. This is the site where tungsten-using extremophilic bacteria (archaea) were found. In soils, the oxidation state of molybdenum varies from II to VI, but only the soluble molybdate anion is available for bacteria (and plants).

In order to gain biological activity, Mo has to be complexed by a special cofactor. With the exception of bacterial nitrogenase, all Mo-dependent enzymes (Mo-enzymes) utilize a molybdenum cofactor (Moco) consisting of a mononuclear Mo atom coordinated via a cis-dithiolene moiety to the organic molecule molybdopterin (MPT) at their catalytic site (50). Accordingly, an extreme conservation of the Moco biosynthetic machinery (see below) is observed. More than 50—mostly bacterial—Mo-enzymes are described in nature. In a few eubacteria and in many archaea, Mo is replaced by tungsten bound to the same unique pterin (49). In this chapter we will review how Moco is synthesized and follow it until its insertion into apo-Mo-enzymes. For the tungsten-dependent enzymes, it is assumed that the formation of the W cofactor follows the same principles as outlined for bacterial Moco (see a recent review, reference 134). At the same time, very restrictive reactions should enable a clear discrimination between these related metals because they are both bioavailable and known to often represent antagonists for each other.

MOLYBDENUM COFACTOR BIOSYNTHESIS IN ESCHERICHIA COLI

Genetics of Molybdenum Cofactor Biosynthesis

The investigation of Mo metabolism started with the genetic analysis of mutants of the filamentous fungus Aspergillus nidulans (106) that were defective in nitrate reductase. Cove and Pateman (26) isolated nitrate reductase-deficient mutants that showed a simultaneous loss of two Mo-dependent enzymes, nitrate reductase and xanthine dehydrogenase. As Mo was the only link between these two otherwise very different enzymes, it was suggested that both enzymes should share a common Mo-related cofactor, named Moco. Later, Johnson et al. (58) demonstrated that the organic compound of Moco from different Mo-enzymes is a unique pterin, which they called molybdopterin (MPT).

In parallel to the achievements of fungal biochemical genetics, also in E. coli, Moco mutants had been isolated by using the same selection principle: growth of mutagenized cells in the presence of high concentrations of chlorate. Mutants selected for chlorate resistance (chl) do not reduce chlorate to the toxic chlorite because they have lost chlorate reductase activity, which appears to be a nonphysiological catalytic activity of the Moco-dependent nitrate reductase (108, 109). The chlorate-resistant phenotype reflects the lack of nitrate reductase activity either due to a mutation in the corresponding structural genes in E. coli or to a loss of Moco. Noteworthy, most of the Moco-dependent enzymes are involved in multiple anaerobic respiratory pathways (see below) and none of them are essential for the growth of E. coli. Accordingly, Moco deficiency is nonlethal, allowing isolation of pleiotropic mutants. It turned out that the loci chlA, chlB, chlD, chlE, and chlG were all essential for Moco biosynthesis, and Stewart and MacGregor (143) isolated a large series of Mu-phage insertion mutants for all these loci. In 1992, the Moco-specific chl loci were renamed in mo loci (138).

Already in pregenomic times, a detailed mutant characterization contributed significantly to our understanding of the genetics and biochemistry of Moco biosynthesis in bacteria, plants, fungi, and humans. Investigations such as phenotype suppression by external molybdate or reconstitution experiments mixing cell-free protein extracts of different complementation groups provided evidence for two intermediates of the biosynthetic pathway. As a next step, defects in molybdate uptake and processing could be assigned to specific mutants. As in all organisms, several different genetic complementation groups were identified, and the existence of a conserved multistep biosynthetic pathway of Moco was proposed. Comprehensive analyses of these mutants involving molecular, genetic, and biochemical studies by several laboratories led to a detailed picture of Moco biosynthesis in E. coli. Five operons (moaABCDE, mobAB, modABCD, modEF, moeAB, and mogA) are required for Moco biosynthesis encoding 16 proteins, and details for these genes/proteins are summarized in Table 1.

Table 1Genetic and biochemical characteristics of the proteins involved in Moco biosynthesis in E. coli

Molybdenum Uptake

When we follow the way that Mo takes from entry into the cell until its final position within the Mo-enzyme's catalytic center, the first step is the active uptake of Mo in the form of its molybdate anion. The analysis of Moco biosynthesis started with the identification of mutants exhibiting a so-called molybdate-repairable phenotype (33). Those mutants were found in all organisms where Moco deficiency was studied, and they are characterized as mutants with partially or completely restored Mo-enzyme activity after growth on unphysiologically high concentrations of molybdate (up to 1 mM). Mo uptake requires specific systems to scavenge molybdate in the presence of competing anions. E. coli cells grown aerobically in 10 nM molybdate contain 1 μM molybdate (107, 136). In E. coli and other bacteria, high-affinity ABC-type molybdate transporters (encoded by the modABCD operon) are described, consisting of three protein components and requiring ATP hydrolysis for operation (reviewed in reference 107). The periplasmic molybdate-binding protein ModA specifically binds molybdate and tungstate with a very high affinity (K_D, 20 to 50 nM). ModB is the dimeric membrane-integral translocation component, and ModC is the cytoplasmic membrane-associated protein that couples ATP hydrolysis with molybdate translocation through the membrane into the cytoplasm (Table 1). The function of ModD is unknown. Diverging from the E. coli modABCD operon is another one encoding ModE and ModF proteins (34). ModF is homologous to ModC and has no defined function. ModE is a transcriptional regulator member of the LysR family present in a few bacteria in which it is found in the molybdate transport locus. The ModE regulator protein comprises two domains, a DNA-binding domain and a molybdate-binding domain (3, 40) with a K_D of 0.8 μM for molybdate. Binding of molybdate increases the ModE affinity for DNA about 25 times due to extensive conformational changes within the molecule (132). The molybdate-bound form of the ModE protein activates transcription of genes for several molybdoenzymes, dimethyl sulfoxide reductase (dmsABC) (91), formate hydrogenlyase (hyc and fdhF), and nitrate reductase (narGHJI) (137), and for molybdenum cofactor biosynthesis (moaABCD) (2, 92) (see "Regulation of Molybdenum Cofactor Biosynthesis," below). While ModA proteins cannot discriminate between molybdate and tungstate, tungsten-specific transporters have been identified in Eubacterium acidaminophilum or in Pyrococcus furiosus (14, 86). In addition to the high-affinity transport system, a low-affinity transport also is operating for molybdate and tungstate, as both anions are cotransported with the sulfate transport system (35).

Regulation of Molybdenum Cofactor Biosynthesis

In E. coli, Moco biosynthesis is enhanced under anaerobiosis already through the moa operon, which encodes the enzymes required for the first step of Moco synthesis. Expression of moa is enhanced under anaerobic growth conditions but is repressed in strains able to synthesize active molybdenum cofactor (7). Molybdate acts as a major positive regulator of moa, and its action requires the ModE protein (2). Transcription of moa is controlled at two sigma-70-type promoters immediately upstream of the moaA gene. The distal promoter is the site of the anaerobic enhancement, which is Fnr dependent. The molybdate induction of moa is exerted at the proximal promoter. The molybdate activation of moa, however, is revealed only in a molybdenum cofactor-deficient background, since moa is effectively repressed in molybdenum cofactor-sufficient strains. Interestingly, tungstate is also able to relieve the repression of the moa operon (2) so that one has to assume that there is a step in Moco formation that can discriminate between active Mo cofactor and functionally inactive W cofactor. (E. coli has no W-dependent enzymes, and so it has no use for W cofactor.) The Mo-MPT-dependent formation of the dinucleotide form of Moco is the most likely candidate for this regulatory link, but no mechanistic details have yet been proposed.

Also, the moe operon is regulated and it was found that its expression is independent of genes coding for Mo transport and for MPT synthesis; instead, anaerobic conditions as well as nitrate were stimulating moe expression (46). Earlier the authors had published that the product of the MoeA-catalyzed reaction is required for Mo-dependent control of genes coding for E. coli Mo-enzymes (45). Apparently, the bacterium coordinates Moco biosynthesis with apoprotein synthesis at the level of moe operon transcription. Finally, the mob locus appears to be constitutively expressed (54).

Biosynthesis of the Molybdenum Cofactor

Most of our knowledge about Moco biosynthesis was obtained from studies in E. coli, and this work was pioneered by Rajagopalan, Johnson, and coworkers (114, 115). Moco biosynthesis proceeds in four steps, and these steps are defined by the following biosynthetic intermediates: cyclic pyranopterin monophosphate (cPMP, formerly precursor Z), molybdopterin (MPT), adenylated molybdopterin, and Mo-bound molybdopterin (Fig. 1). In prokaryotes, during a fifth step a nucleotide is added, thus forming the Mo-bisMGD (however, we will describe steps four and five as one combined last step) (Fig. 1).

Fig. 1Molybdenum cofactor biosynthesis in E. coli. Shown are the known biosynthetic intermediates dividing the whole pathway in four steps. Ribbon representations of the crystal structures of the Moco biosynthetic proteins are shown: MoaA (43), MoaC (159), MoaD-MoaE complex (122), MoeB-MoaD complex (66), MogA (77), MoeA (163), MobA (65, 141), and MobB (90). Individual figures were generated with PYMOL (28) using the deposited coordinates from the protein structure data base.

Step 1: Conversion of GTP to cPMP.

MPT is the only pterin known to be substituted with a four-carbon side chain, while several other pteridines such as biopterin have three-carbon side chains. Two pathways are known for the synthesis of pteridines (147) and flavins (5) that start with the conversion of GTP by the enzymes cyclohydrolase I and II, respectively, whereas Moco synthesis depends on a third route also starting with GTP. Based on labeling studies in E. coli, Wuebbens and Rajagopalan (160) revealed a complex reaction sequence starting from GTP (43) and generating the first stable intermediate of Moco biosynthesis, cPMP. Originally, this first intermediate was named precursor Z (162). Recently, its chemical structure was clarified by mass spectrometry and ¹H NMR spectroscopy (125). It was demonstrated that the molecule is a pyranopterin, similar to Moco, and carries a geminal diol at the C1' position of the side chain. Therefore, precursor Z was renamed cPMP for cyclic pyranopterin monophosphate (125).

In E. coli, two proteins, MoaA and MoaC, were identified as essential for cPMP synthesis (Fig. 1 and Table 1). MoaA contains two oxygen-sensitive [Fe-S] clusters that are bound via three highly conserved cysteine residues (41) and shows sequence similarities to a number of proteins, including biotin synthase or thiamine synthase. MoaA and all homologues belong to the family of S-adenosylmethionine (SAM)-dependent radical enzymes. Members of this large family catalyze the formation of protein and/or substrate radicals by reductive cleavage of SAM by a [4Fe-4S] cluster (68, 140). The structure of MoaA was determined in the apo- state as well as with the cosubstrate SAM or the 5'-GTP (42, 43). These data were of considerable value because they provided insights into the radical reaction underlying the conversion of 5'-GTP to cPMP. Indeed, MoaA is not able itself to catalyze the release of pyrophosphate, which indicates that, during catalysis, 5'-GTP and its reactive radical intermediates are tightly anchored by the triphosphate moiety to prevent their escape. MoaC appears to be responsible for pyrophosphate release. The X-ray crystallographic structure of E. coli MoaC reveals that it forms a homohexamer (159) with a hypothetical active site formed by several strictly conserved residues at the interface of two MoaC monomers. Nevertheless, complete understanding of this reaction step, which most likely involves multistep reactions, must await further studies. Also the detailed function played by MoaC remains enigmatic.

Step 2: Conversion of cPMP to MPT.

During the second step of Moco biosynthesis, the MPT dithiolate is formed by incorporating two sulfur atoms into cPMP (Fig. 1 and 2). This reaction is catalyzed by MPT synthase, a heterotetrameric complex of two small (MoaD) and two large subunits (MoaE) that stoichiometrically converts cPMP into MPT (Fig. 2 and Table 1). Biochemical studies using in vitro assembled MPT synthase from individually expressed and purified subunits demonstrated that the C terminus of MoaD carries the sulfur as thiocarboxylate (39). The functional importance of this thiocarboxylate was also demonstrated in the crystal structure of the E. coli MPT synthase, which shows that the C terminus of MoaD is deeply inserted into the large subunit MoaE to form the active site (122). The heterotetramer is formed by dimerization of two large subunits forming two clearly separated active sites. Interestingly, although the thiocarboxylate moiety on the MoaD subunit is essential for MPT synthase activity, it is not required for formation of the synthase heterotetramer. Detailed investigations of the thermodynamic properties of the interaction between MoaD and MoaE in MPT synthase revealed an increased binding affinity of MoaD-SH to MoaE consistent with the proposed reaction mechanism (148). Moreover, the solvent-accessible surface area buried on formation of the heterotetramer was considerably increased on activation of the protein, changing from 2,376Å to 4,117Å. Because each small subunit of MPT synthase carries a single sulfur atom, a two-step mechanism for the formation of MPT dithiolate has been proposed, which involves the formation of a monosulfurated intermediate (39, 161).

Fig. 2Conversion of cPMP to MPT (step 2). See the text for a detailed description of the reaction mechanism leading to the two-step conversion of cPMP to MPT.

After MPT synthase has transferred the two sulfurs to cPMP, it has to be resulfurated in a separate reaction by one or more proteins. This resulfuration is catalyzed by MoeB involving an adenylation of the small subunit MoaD in a MoaD-MoeB complex by using Mg²⁺-ATP as substrate followed by sulfur transfer (76) (Fig. 2). The crystal structures of MoeB in complex with MoaD have been determined in the apo-, ATP-bound, and MoaD-adenylate form (66), presenting a conserved mechanism of acyl-adenylate formation in ubiquitin-dependent protein degradation and in the synthesis of Moco. MoaD and homologous proteins harbor in their C-terminal region a conserved double-glycine motif also found in ubiquitin, a crucial protein in eukaryotic protein degradation (48). Only the terminal glycine appears to be essential for proper function of MoaD (131). Besides the homology of MoaD and MoeB to the ubiquitin-activating system, similarities between Moco biosynthesis and thiamin biosynthesis can also be seen in E. coli. The proteins ThiF, ThiS, and ThiI participate in the synthesis of the thiazole moiety. Begley and coworkers (69, 144, 156) have shown that ThiS is thiocarboxylated by ThiF (= homologous to MoeB) and ThiI (= sulfur transferase). A similar way of sulfur activation (i.e., adenylation of the sulfur transfer protein MoaD or ThiS followed by exchange of AMP for sulfur) has been proposed for the synthesis of biotin (9) and lipoic acid (25) (for review, see reference 60).

MoeB alone is not sufficient to reactivate carboxylated MoaD. Free sulfide or persulfide-loaded cysteine desulfurases, such as IscS, SufS, or CsdA, are needed to cleave the acyl-adenylate of MoaD, thus forming thiocarboxylated MoaD that is able to assemble into an active MPT synthase with MoaE (75) (Fig. 2). Despite the fact that, in vitro, SufS appears to be the preferred one among the three cysteine desulfurases, the in vivo sulfur source remains unknown. A redundant function of different persulfide-generating systems is possible (75).

Step 3: Adenylylation of MPT.

After synthesis of MPT, the chemical backbone is ready to bind and coordinate the molybdenum atom. Mo has to be taken up into the cell in the form of molybdate, followed by the coordination to MPT. Once inside the cell, a key question of Moco biosynthesis resides in whether the molybdate serves as a donor for insertion of Mo into MPT or whether it has to undergo intracellular processing prior to insertion. Below, we will discuss the existence of an additional intermediate in Moco biosynthesis, adenylated MPT (MPT-AMP), preceding the Mo insertion step (Fig. 3).

Fig. 3Adenylylation of MPT (step 3). See the text for a description of the reaction mechanism leading to MPT adenylylation.

In bacteria, two proteins MogA and MoeA are involved in Mo insertion (Table 1), while during evolution to higher organisms, these two proteins were fused to a two-domain protein. Whereas it had been early postulated that one protein should be essential for MPT binding, the other being in charge of generating an activated form of Mo, the exact mechanism has only been recently uncovered in plants where the protein Cnx1 is catalyzing this step (79). The C-terminal domain (Cnx1-G) known to complement a mogA mutant was shown to tightly bind MPT (133). Crystal structure of Cnx1-G in complex with MPT confirmed the proposed binding of MPT (63). Further, the Cnx1-G active site was mapped by structure-based mutagenesis and functional analysis to a large surface depression with a clear discrimination between MPT binding and catalysis (64, 135). Unexpectedly, the structure of a variant (S583A) with a gain of function revealed a novel intermediate in Moco biosynthesis as an adenosine moiety was covalently bound via a pyrophosphate bound to the C4' carbon of MPT, thereby forming adenylated MPT (63). Subsequently, it has been demonstrated that Cnx1-G adenylates MPT in a Mg²⁺- and ATP-dependent way and forms MPT-AMP that remains bound to Cnx1-G (79) (Fig. 3). This intermediate appears to be mechanistically relevant because it serves as substrate for the subsequent Mg²⁺-dependent Mo insertion reaction by MoeA (or the equivalent Cnx1-E domain) (80) (Fig. 4). Based on the ability of Cnx1-G to reconstitute mogA mutants and on their nearly identical X-ray structures, one can conclude that both proteins catalyze the MPT adenylation reaction, which is essential for and takes place prior to metal insertion.

Fig. 4Mo insertion and nucleotide addition (step 4). See the text for a description of the reaction mechanism leading to Mo addition and of the different postulated pathways for the nucleotide addition step leading to the Mo-bisMGD molecule.

Within the moa operon, the second open reading frame encodes for a protein, MoaB, with significant homologies to MogA (Table 1). Crystal structures of MoaB (6, 124) and MogA (77) confirmed their strong structural similarity, supporting the idea of a conserved function. However, the function played by MoaB remains enigmatic because mogA mutants show Moco deficiency, while moaB mutants did not affect the activity of Mo-dependent enzymes in E. coli (I. Lueke, G. Schwarz, R. R. Mendel, and T. Palmer, unpublished results). Careful examination of the MoaB structure reveals the absence of the catalytically essential residues present in MogA, pointing toward a loss of function of MoaB in E. coli. In archaea, MoaB proteins could have a MogA-like function in the biosynthesis of W-cofactors since MoaB homologous proteins were predominantly found (134).

Step 4: Mo Insertion and Nucleotide Addition.

Considering that adenylated MPT is an intermediate of Moco biosynthesis in bacteria as well, two further steps need to be accomplished for synthesis of the active form of Moco found in most prokaryotic Mo-enzymes. Indeed, the vast majority of these enzymes that make use of Mo do so through nucleotide-substituted Moco. In E. coli, this cofactor contains a nucleotide, a guanosine monophosphate, covalently linked to MPT via a pyrophosphate bond resulting in the MPT dinucleotide cofactor (Fig. 1). Other prokaryotic variants of the cofactor containing CMP, AMP, or IMP linked to the MPT were identified as well (114). It is important to notice that, in addition to the nucleotide moiety, another difference compared with eukaryotes is the formation of bis-MPT-based cofactors where one Mo (or W) atom is coordinated by two dithiolenes of two MPT molecules. Therefore, in addition to Mo insertion, dinucleotide formation has to be performed in the final stages of Moco biosynthesis. There is cumulative evidence that these two steps are linked to each other, so that we will discuss both steps as a fourth step of Moco biosynthesis.

For a long time, the exact mechanism underlying Mo incorporation remained one of the most enigmatic aspects of Moco biosynthesis. Leimkühler et al. (70) reported that the activity of the MPT-dependent xanthine dehydrogenase in a Rhodobacter capsulatus moeA mutant could be recovered on growth with 1 mM sodium molybdate, indicating that MoeA is involved in Mo incorporation. Later on, Nichols and Rajagopalan (96) clearly demonstrated that, while mutations in either mogA or moeA have no effect on MPT biosynthesis, they completely abolish the ligation of Mo to MPT. Nevertheless, the vast majority of E. coli Mo-enzymes contain a bis-MPT-type cofactor and moeA mutants show no molybdate-repairable phenotype with respect to the activity of those enzymes (44). One can argue that a bis-MPT-based cofactor should require a different metal insertion process than a mono MPT-based cofactor, as found in all eukaryotes as well as in some bacterial enzymes. Interestingly, a novel MPT-type oxidoreductase (YedY) was identified and characterized in E. coli that belongs to the eukaryotic sulfite oxidase family of Mo-enzymes (21, 81). These data indicate that both mono- and bis-MPT forms of Moco do exist in E. coli. In this context, it is important to notice that the function of E. coli moeA cannot be reconstituted by Cnx1-E as seen on the basis of bis-MPT-dependent enzyme activity. These observations provide further support for functional diversity between bacterial and eukaryotic Mo insertion processes. In vitro studies have shown that MogA stimulates, in an ATP-dependent manner, the activity played by MoeA in mediating Mo incorporation to MPT using eukaryotic MPT-dependent aposulfite oxidase as reporter enzyme (97). Future investigations aiming at testing the effect of these purified proteins on the activity of a bis-MPT-type cofactor-dependent enzyme are thus of substantial importance.

Due to its intrinsic instability, Moco has to remain bound to proteins during the whole biosynthetic process until its final delivery to apo-Mo-enzymes. Interestingly, the use of an in vivo approach, the bacterial two-hybrid system, was proven to be valuable in determining the conditions required for visualization of the interaction between proteins involved in the late stages of Moco biosynthesis (Fig. 5). Indeed, MPT appears to be of crucial importance for the interaction between MogA and MoeA (83). Based on the conserved fusion event occurring between eukaryotic MogA and MoeA and on the observed interactions between E. coli counterparts in the presence of MPT, Magalon et al. (83) suggested that during evolution it became important to facilitate substrate-product flow by the existence of a Moco-biosynthetic multienzyme complex. Formation of such complexes would ensure both the fast and protected transfer of reactive and oxygen-sensitive intermediates within the reaction sequence from MPT to Mo-MPT. These data pointed to a concerted mechanistic action of MogA and MoeA, and this concept was supported by biochemical analyses performed by Llamas et al. (79, 80), as seen below. Earlier biochemical studies had indicated that newly formed MPT remains tightly bound to the MPT synthase complex until its transfer to MogA by direct protein interaction (74). The same applies to the newly synthesized adenylated MPT remaining associated with Cnx1-G (equivalent to MogA) (79). Later on, Llamas et al. (80) demonstrated that MPT-AMP and molybdate bind with high affinity in a cooperative and equimolar manner to Cnx1E. Once transferred from Cnx1G to Cnx1E, MPT-AMP is rapidly hydrolyzed in the presence of Mg²⁺ or Zn²⁺ with rates that are several orders of magnitude higher than MPT-AMP synthesis (79) (Fig. 4). Therefore, MPT-AMP synthesis seems to be the rate-limiting step in Cnx1 reaction. Further, these authors have shown that MPT-AMP hydrolysis resulted in stoichiometric release of Mo-MPT that was quantitatively incorporated into plant aposulfite oxidase. This reaction mechanism established for plants seems to hold true also for E. coli where a mutational analysis recently carried out for MoeA suggested functions similar to the plant protein (98). In summary, MogA and MoeA are both essential for the two-step reaction leading to metal transfer to MPT.

Fig. 5Interactions network among Moco biosynthetic proteins in E. coli. The arrows represent the interactions as detected by using bacterial two-hybrid methodology, TAP-Tag, or biochemical assays (see the text for details and references).

While the reaction mechanism that leads to the formation of Mo-MPT is known, in which order, however, must those steps take place that lead not only to nucleotide addition, but also to formation of a bis-MPT-type cofactor? At first, the mobAB locus is responsible for nucleotide attachment in E. coli Moco biosynthesis (Fig. 4 and Table 1). MobA catalyzes the conversion of MPT and GTP to MGD (104), whereas MobB, a GTP-binding protein, is not absolutely required for MGD synthesis (54, 102). Using a fully defined in vitro system, Temple and Rajagopalan (146) demonstrated that MobA alone, when incubated with GTP, Mg²⁺, and a source of MPT catalyzes the formation of MGD, indicating that it is both necessary and sufficient for GMP attachment. Specific protein-protein interactions have already been shown to play a central role in the early stages of Moco biosynthesis (117). In the same way, Magalon et al. (83), with the use of a bacterial two-hybrid approach, were able to demonstrate that MobA interacts with MoeA and MobB in vivo (Fig. 5). In particular, the interaction between MobA and MoeA strictly depends on the presence of MPT. The crystal structure of MobA was solved and indicated an overall α/β architecture and a nucleotide-binding Rossman fold within the N-terminal half (65, 141). The active site was defined by highly conserved residues as well as by cocrystallization of MobA with GTP that is bound in the N-terminal half (65). An important finding was that MobA can also be copurified along with MPT and MGD, demonstrating a tight binding of both its substrate and product (38).

Consistent with its ability to bind GTP, the amino acid sequence of MobB reveals a putative nucleotide-binding motif, the Walker A motif. Crystal structure of MobB indicated a dimeric state of the protein and confirmed the lack of structural elements required to interact with and efficiently bind a nucleotide base (90). Structural homologues of MobB include a number of nucleotide-binding proteins. Based on the observation that MobA and MobB interact in vivo, McLuskey et al. (90) proposed a model in which the formation of a MobA-MobB complex enhances the efficiency of conversion of MPT to MGD through better GTP binding and utilization.

Interestingly, MobB interacts not only with MobA but also with MogA and MoeA (83) (Fig. 5). The functional signification for such interactions is not yet understood. It is worth mentioning that MobB does exist in some organisms as a fusion protein with MoeA in gamma-proteobacteria such as Vibrio species and Shewanella oneidensis (Fig. 6). In this case, MoeA exists in two forms, fused with MobA or not, both forms harboring the essential catalytic residues. In other bacteria such as Chlorobium species or Geobacter metallireducens, MobB is fused with MobA. An extensive network of protein interactions has thus been revealed among proteins involved in the final stages of Moco biosynthesis. Although clear understanding of these steps has not yet been attained, these data provide further evidence that the processes of Mo insertion and of dinucleotide attachment are strongly linked. Clearly, understanding of the exact function played by MobB in Moco biosynthesis awaits further studies.

Fig. 6Schematic representation of the fusion proteins involving MobB. Identity percentages are indicated by using E. coli proteins as a reference.

ASSEMBLY OF MO-ENZYMES AND INSERTION OF MOCO

After synthesis and maturation, Moco has to be incorporated into the appropriate apoenzymes. Because Moco is labile and oxygen sensitive, it was assumed that there is no free Moco occurring in the cell, and it was suggested that Moco should be transferred immediately after biosynthesis to the apoenzyme or that it could be bound to a carrier protein that protects and stores Moco until further use. Such proteins were described for Rhodospirillum rubrum (61), E. coli (1), and Chlamydomonas reinhardtii (158). The initial evidence for the existence of a Moco-synthetic machinery came from studies with E. coli, where Magalon et al. (83) used a two-hybrid approach to identify an extensive network of interaction between proteins catalyzing the final steps of Moco biosynthesis. By using the same method, further interactions between these proteins and aponitrate reductase were described that might point to a direct transfer of Mo-bisMGD from the machinery to apoenzymes (154). In the case of the nitrate reductase, an enzyme-specific chaperone NarJ is further required for Moco insertion as seen by its absolute necessity for mediating the interaction between the Moco delivery machinery and the apoenzyme. Other system-specific chaperones have also been reported to be involved in Mo-enzyme maturation (see "Discovery of Enzyme-Specific Chaperones," below). In summary, Moco transfer to prokaryotic Mo-enzymes is ensured by the proteins involved in the final stages of Moco biosynthesis and assisted, in some cases, by enzyme-specific chaperones.

Another consideration is that in all known Mo-enzymes, the crystal structure reveals that Mo-bisMGD is an extended molecule (35 Å) deeply buried in the enzyme which, in some cases, is in close proximity to other redox centers or at subunit interface. This observation suggests that Moco insertion is intimately connected to protein folding and subunit assembly.

To decipher the Moco incorporation step, several groups used in vitro reconstitution assays of Mo-enzymes activity. The use of Rhodospirillum sphaeroides dimethyl sulfoxide (DMSO) reductase (146) or of E. coli trimethylamine N-oxide (TMAO) reductase (52) in reconstitution assays was made to eliminate many of the complications associated with nitrate reductase biogenesis (16, 102). Indeed, these enzymes are single soluble subunits that contain Mo-bisMGD as the sole prosthetic group (27, 128), whereas nitrate reductase is a membrane-bound, heterotrimeric complex that contains five [Fe-S] clusters and two heme groups in addition to the Moco (13, 59). Completely defined in vitro systems for studying the mechanism of Moco insertion were thus established for both the DMSO and TMAO reductases. The main conclusion is that maximal activation requires a long incubation period exceeding several hours. Thus, it is likely that several factors are missing under these conditions; one of them is the multiprotein complex of Moco biosynthetic proteins in charge of Moco delivery to the apoenzyme. Notably, additional presence of TorD, the TMAO reductase-specific chaperone, considerably increased the level of activation while still requiring a longer incubation period (52). Interestingly, in the case of the complex nitrate reductase system, NarJ is absolutely required for Moco incorporation (16, 102).

Discovery of Enzyme-Specific Chaperones

Early work in Giordano’s group has shown that a lesion in the narJ gene present in the nar operon, encoding the nitrate reductase A complex, blocks the generation of an active enzyme complex (19). Similarly, the fdhD and fdhE genes flanking the fdo operon encoding the aerobic formate dehydrogenase-O complex in E. coli are necessary for synthesis of both active formate dehydrogenase-O and -N (129, 130). Due to its essential character, the NarJ protein has constituted the prototype of an accessory protein for Mo-enzymes in prokaryotes. Later on, other groups reported the implication of similar proteins in the synthesis of active Mo-enzymes, such as TMAO reductase (111), DMSO reductase (101, 116), or xanthine dehydrogenase (73). This list considerably expanded with the genomic era, and numerous operons coding for prokaryotic Mo-enzymes have revealed the existence of additional genes, each encoding a putative system-specific chaperone (10, 62, 89, 113).

In the following we will discuss three types of chaperones representing four different cases of Mo-enzyme assembly and Moco insertion: (i) NarJ as an example for a cytoplasmic and multimeric Mo-enzyme, (ii) TorD as an example for a periplasmic and monomeric Mo-enzyme, (iii) DmsD and FdhD/FdhE as examples for periplasmic and multimeric Mo-enzymes, and (iv) XdhC as an example for a Mo-MPT enzyme.

Cytoplasmic Multimeric Mo-Enzymes and NarJ

The E. coli dissimilatory quinol-nitrate oxidoreductase of the anaerobic respiratory chain, referred to as the nitrate reductase A (NarGHI) (17, 119), constituted one of the first studied Mo-enzymes in terms of maturation pathway. NarGHI is a nonexported membrane-bound complex composed of three subunits that bind eight redox centers: (i) a catalytic subunit (NarG) containing a Mo-bisMGD cofactor and a proximal [4Fe-4S] cluster (FS0) (13, 59), (ii) an electron transfer subunit (NarH) carrying one [3Fe-4S] cluster (FS4) and three [4Fe-4S] clusters (FS1 to FS3) (36, 37), and (iii) a quinol-oxidizing membrane-bound subunit (NarI) containing two b-type hemes (b_D and b_P) (84, 118, 119) (Fig. 7). Initial biochemical and genetic studies indicated that the NarJ protein encoded by the narGHJI operon plays an essential role in nitrate reductase activity promoting correct assembly of the enzyme complex without being part of the final structure (18, 19, 29). Based on its properties, a role of private or system-specific chaperone has been first proposed (19, 78). E. coli synthesizes a second nitrate reductase complex, the NarZYV isoenzyme, whose maturation involves the NarW protein and a NarJ homologue, both being exchangeable.

Fig. 7Schematic representation of the membrane-bound nitrate reductase from E. coli. (Left) The NarGHI complex is surface-represented (PDB ID code 1q16). The Moco-containing catalytic subunit NarG is shown in yellow, the electron-transfer subunit NarH is shown in orange, and the membrane-bound subunit NarI is shown in blue. (Right) Stick representation of the different metal centers.

A detailed biogenesis model for how sequential interaction between NarJ and the apoenzyme promotes assembly and Moco incorporation within the multisubunit nitrate reductase complex is depicted in Fig. 8. A major advance in understanding the NarJ function came from detailed analyses of the cofactor content of the nitrate reductase complex produced in absence of NarJ revealing a lack of Moco (16). Biochemical studies (154) demonstrated that NarJ enables Moco incorporation through a direct protein interaction with the catalytic subunit NarG. Further, two distinct NarJ-binding sites were mapped on the NarG catalytic subunit by structure-based deletion and functional analysis (155). One as yet undefined site allows the interaction of apoNarGH with the Moco biosynthetic machinery and thus allows Moco insertion (154). Unexpectedly, a second site is formed by the first 40 amino acids of the N-terminal tail of NarG that are responsible for the interaction of NarG with NarI (155), as revealed by the X-ray structure of the NarGHI complex (13). Finally, a short peptide encompassing the first 15 residues of NarG allows NarJ binding with a dissociation constant in the micromolar range (24). Vergnes et al. (155) showed that NarJ binding to this site interferes with membrane anchoring of the apoenzyme complex. Further, once bound to the NarI subunit, the apoenzyme complex cannot sustain NarJ-assisted Moco incorporation, indicating a definitive conformation acquired on membrane anchoring (16, 155). Accordingly, Moco incorporation is a cytoplasmic event that must take place prior to membrane attachment of the apoenzyme (Fig. 8). Further, deletion of the N-terminal tail leads to membrane anchoring of a partly mature enzyme complex that ultimately forms a physiologically inactive nitrate reductase complex (155). These observations supported a role of NarJ in the coordination of subunit assembly and cofactor insertion processes.

Fig. 8Biogenesis model of the E. coli nitrate reductase A (NarGHI) and the TMAO reductase (TorCA) complexes. NarG and NarH constitute the catalytic dimer, while NarI is the b-type membrane anchor subunit of the complex. NarI maturation takes place in the inner membrane where the two b-type b_D and b_P hemes are sequentially inserted. Concomitantly, the apoNarGH complex retained by the enzyme-specific chaperone NarJ in the cytoplasm is maturated. First, [Fe-S] clusters are inserted in the NarH subunit through the action of one of the [Fe-S] biosynthetic machineries. Second, both Moco and its proximal [Fe-S] cluster, FS0, are inserted in the catalytic subunit NarG in a NarJ-dependent manner. On complete maturation of the NarGH complex, NarJ dissociates, allowing membrane anchoring of the NarGH dimer. TorA constitutes the catalytic subunit of the TMAO reductase system and harbors a twin-arginine signal peptide at the N terminus. Early interaction of the enzyme-specific chaperone TorD on apoTorA facilitates Moco insertion. Subsequently, mature TorA is exported to the periplasm through the Tat translocase. TorC, a pentahemic membrane-bound cytochrome c, constitutes the electron donor to TorA. Whatever the considered system, Moco insertion proceeds as the action of a multiprotein complex of Moco biosynthetic proteins and chaperones (70, 125).

An important finding came from a systematic analysis by electron paramagnetic resonance spectroscopy of stable maturation intermediates of the nitrate reductase complex (67). Indeed, a broad defect in metal incorporation has been demonstrated in the absence of the accessory protein NarJ. While the lack of Moco does not preclude the insertion of the proximal [Fe-S] center FS0 within the NarG catalytic subunit, the absence of NarJ leads to the specific loss of both Moco and FS0 from NarG without having any effect on the insertion of the four [Fe-S] centers from NarH (67). Moreover, the presence of FS0 appears to be a prerequisite for Moco insertion. In summary, Moco and FS0 insertion are cytoplasmic events occurring before membrane attachment of the enzyme complex, and NarJ is absolutely required for these events (Fig. 8).

An open question concerns the direct versus indirect role of NarJ in FS0 insertion within the catalytic subunit NarG. In E. coli, formation of [Fe-S] clusters requires complex biosynthetic machineries (Isc, Suf, or CSD) and involves scaffold proteins and chaperones during their insertion step (for review, see references 8 and 57 and Chapter From Iron and Cysteine to Iron-Sulfur Clusters: the Biogenesis Protein Machineries). Two hypotheses can thus be proposed: (i) NarJ binds an [Fe-S] center and delivers it directly to the aponitrate reductase complex, or (ii) NarJ facilitates the action of a scaffold protein in delivering the [Fe-S] cluster. While sequence analysis of NarJ and/or NarW proteins does not reveal any consensus motif for [Fe-S] cluster coordination, it cannot be ruled out that these proteins may transiently accommodate an [Fe-S] through unorthodox coordination.

Assembly of the nitrate reductase complex is a multiple-step process including metal cofactor insertion, subunit assembly, and membrane anchoring. Additionally, Lanciano et al. (67) demonstrated that premature membrane anchoring of the apoenzyme complex in the absence of NarJ leads to incomplete maturation of the NarI subunit with the specific absence of the cytoplasmically oriented b_P heme. NarJ is thus indirectly required for complete maturation of NarI by keeping the NarGH complex in a soluble state until the Moco insertion step. Altogether, these data demonstrated that NarJ orchestrates the cofactor insertion, subunit assembly, and membrane-anchoring steps during the assembly of the nitrate reductase A complex (Fig. 8). Importantly, it can be inferred from comparison with other multimeric Mo-enzymes that the global function played by NarJ in the biogenesis process of the nitrate reductase complex will be extended to other related systems.

Periplasmic Monomeric Mo-Enzymes and TorD

While NarJ can be seen as the prototype of a class of chaperones required for maturation of cytoplasmic Mo-enzymes, how does Moco insertion proceed in the case of Mo-enzymes exported to the periplasm? In the case of the E. coli periplasmic TMAO reductase, Santini et al. (126) demonstrated that Moco insertion is a prerequisite for translocation of the enzyme. Moreover, the export of this kind of folded protein across the bacterial cytoplasmic membrane is ensured by the twin-arginine translocation (Tat) pathway (11, 103, 127). It is also apparent that unfolded proteins are not compatible with transport, implying that there must be steps in which some sort of quality control is exerted on the folding state of the substrate. Cytoplasmic system-specific chaperones that are required for the activity of periplasmic Mo-enzymes in E. coli are likely to be responsible for such control.

In E. coli, reduction of TMAO is ensured mainly by a respiratory system encoded by the inducible torCAD operon (93, 139) (see Chapter S- and N-Oxide Reductases). The terminal reductase, TorA, is a periplasmic Mo-enzyme of 97 kDa harboring Mo-bisMGD cofactor as the sole prosthetic group as disclosed by the X-ray structure of the Shewanella massilia counterpart (27). The last gene of the tor operon, torD, encodes a cytoplasmic protein of 23 kDa behaving as a private chaperone of TorA (111). Pommier et al. (111) reported that a torD knockout mutant produces twofold less but still active and correctly localized TorA. Further analysis revealed a direct protein interaction between TorD and either apo- or holo-TorA (111, 150). Interestingly, the TorD chaperone from S. massilia forms multiple and stable oligomeric species and crystallized as a dimer (149, 150). The X-ray structure of the protein (PDB ID code 1N1C) reveals extreme domain swapping between the two monomers, providing further evidence for high flexibility of the TorD protein (149). Nevertheless, the biological significance of these oligomers is still under debate as no gain of function in terms of binding affinity, usually encountered through domain swapping (88, 112, 145), has been observed on dimerization. Finally, the TorD dimer displays an all-helical architecture allowing the description of a novel protein fold for enzyme-specific chaperones (149) (Fig. 9).

Fig. 9Space-filling structure of different molybdoenzyme-specific chaperones. (A) NarJ from Archaeoglobus fulgidus (PDB ID code 2o9x). (B) TorD dimer from Shewanella massilia (PDB ID code 1n1c). (C) DmsD from Salmonella enterica serovar Typhimurium LT2 (PDB ID code 1s9u). (D) NapD from E. coli (PDB ID code 2jsx). (E) FdhE from Pseudomonas aeruginosa (PDB ID code 2fiy). Individual figures were generated with PYMOL (28) by using the deposited coordinates from the protein structure database.

Using an in vitro system, Ilbert et al. (52) demonstrated that Moco insertion within apoTorA is strongly facilitated by the presence of TorD and that preincubation of apoTorA with TorD significantly increases the level of in vitro Moco insertion, pointing to a folding action of TorD. In summary, TorD binding to a yet undefined binding site of TorA renders it competent for Moco insertion through folding processes. Additional evidence for a folding activity of TorD on its partner TorA came from studies performed at high temperatures, highlighting the essential character of TorD under these extreme conditions (31).

Considering the periplasmic location of the TorA enzyme and the fact that Moco incorporation is a strictly cytoplasmic event, it is clear that the TorD action enhances considerably the level of correctly folded TorA protein amenable for Tat translocation. Nevertheless, it is important that export of cofactorless Tat substrates like apoTorA is not performed until all assembly processes are complete. Several authors proposed that mechanisms are likely to exist to ensure proper coordination between cofactor insertion and export processes (12, 126). One of the mechanisms is based on the idea that signal peptides of Tat precursors are prevented from interacting with the Tat translocase until cofactor insertion into the precursor protein is accomplished. Jack et al. (56) demonstrated that TorD recognizes the TorA twin-arginine signal peptide as DmsD does it for DmsA (101). Altogether, these data point toward the existence of at least two distinct TorD binding sites on the TorA protein: one is located within the Tat signal peptide, while the other is within the remaining "mature" portion of the protein (53, 56) (Fig. 8). Deletion of the signal peptide has no significant effect on the TorD function as judged by the TorA activity (53). Interestingly, Hatzixanthis et al. (47) reported that TorD has a weak affinity for GTP, which is enhanced by signal peptide binding. This observation led the authors to propose that nucleotide binding and release might regulate the interaction between the signal peptide and TorD. Indeed, it is important that, after completion of the maturation process of the TorA protein, TorD dissociates from the signal peptide.

Recently, Genest et al. (32) reported TorA signal peptide protection by TorD regardless of the presence of Moco and or the Tat translocase. At this stage, it is not clear whether TorD binding to the signal peptide monitors the folding and assembly of the substrate, or alternatively, retards the export kinetics sufficiently to allow completion of the Moco insertion process.

Periplasmic Multimeric Mo-Enzymes and DmsD or FdhD/FdhE

Importantly, most of the periplasmic Mo-enzymes are composed of several subunits and harbor many metal cofactors. Biogenesis of these complexes is an intricate process that requires several steps, such as the synthesis of the different subunits, their assembly, the incorporation of various types of metal cofactors, and the translocation of the catalytic dimer. Although it is most likely that all these events occur in a coordinate fashion to finally yield a functional multimeric Mo-enzyme, information about how this coordination is performed is scarce. Two examples will be provided here with the DMSO reductase (DmsABC) and the formate dehydrogenase (FdnGHI), two periplasmically oriented and membrane-bound heterotrimeric complexes sharing strong similarities in terms of subunit and redox cofactor composition with the nitrate reductase complex. Consequently, biogenesis of these periplasmic and multimeric Mo-enzymes could probably follow the same pathway as outlined for nitrate reductase and be assisted by an enzyme-specific chaperone sharing functional similarities to NarJ.

E. coli DMSO reductase is composed of three subunits: (i) a catalytic subunit (DmsA) containing a Mo-bisMGD cofactor (15, 120) and a putative [4Fe-4S] cluster (151), (ii) an electron transfer subunit (DmsB) carrying four [4Fe-4S] clusters (23), and (iii) a quinol-oxidizing membrane-bound subunit (DmsC) (30, 121, 157) (see Chapter S- and N-Oxide Reductases). Oresnik et al. (101) reported that DmsD is essential for the assembly of a fully active DmsABC complex. The dmsD gene (formerly ynfI) is part of the ynf operon encoding a cryptic DMSO reductase homologue (82, 101). Further, DmsD was isolated as a protein binding the Tat signal peptide of both TorA and DmsA (101). Finally, deletion of the DmsA signal peptide results in the formation of a less stable but soluble and cytoplasmically active DmsAB complex (123). Accordingly, the DmsA variant has likely benefited from the action of DmsD to a second unidentified binding site. Concerted action of DmsD on both the signal peptide and most likely to a second site of the DmsA protein, in the same way as NarJ or TorD, is thus required for productive synthesis of DmsABC. An alternative model for DmsD is based on its ability to interact with two components of the Tat translocase, namely TatB and TatC located in the inner membrane, indicating that it may have a direct role in Tat-mediated export (105).

E. coli synthesizes two respiratory formate dehydrogenase (FDH) complexes, namely, the nitrate-inducible FDH-N (FdnGHI) and the constitutively expressed cryptic FDH-O (FdoGHI). Genetic studies demonstrated that both fdhD and fdhE gene products located astride the fdo operon are involved in the formation of active FDH enzymes (87, 110, 129, 142). FdhD (30 kDa) contains several cys residues and displays a mostly helical architecture (153). FdhE (34.5 kDa) possesses four conserved CX₂C motifs, suggesting metal-binding ability. The recently solved crystal structure at 2.1Å of FdhE in Pseudomonas aeruginosa (PDB ID code 2fiy) revealed that each two pairs of CX₂C motifs located in disordered loops coordinate an iron atom (Fig. 9). While the functional significance of such iron binding remains to be proven, it appears to contribute significantly to the overall folding of the protein. Interestingly, Butland et al. (22) reported the interaction between FdhD and the cysteine desulfurase IscS, a key player in [Fe-S] cluster biosynthesis or selenocysteine incorporation within the catalytic subunit of FDHs. As a consequence, it can be speculated that this interaction is important for selenocysteine and/or [Fe-S] incorporation into FDHs. Further, the interaction between FdhE and FdnG, the catalytic subunit of the FDH-N complex was reported, pointing toward a Tat signal peptide-binding function (22).

E. coli synthesizes a third formate dehydrogenase, FDH-H (FdhF), composed of a single cytoplasmic subunit and harboring a [4Fe-4S] cluster in addition to the Mo-bisMGD cofactor (20). Until now, no enzyme-specific chaperones have been reported to be involved in biogenesis of this Mo-enzyme. Considering its relatedness to the FdnG/FdoG subunits, it is likely that FdhF also requires a protein, ensuring sequential and coordinate incorporation of both the [Fe-S] cluster and the Moco in a NarJ-like manner.

Similarly, maturation of the E. coli periplasmic nitrate reductase complex involves two accessory proteins, NapD and NapF, the latter being proposed to be involved in [Fe-S] cluster insertion within the catalytic subunit NapA (99, 100), while the former is involved in Tat signal peptide binding (85). Recently, Maillard et al. (85) reported the NMR structure of NapD in E. coli displaying a ferredoxin-type fold in contrast to all other proteins having a twin-arginine signal-peptide-binding function such as DmsD or TorD (Fig. 9).

Mo-MPT Containing Mo-Enzymes and XdhC

While E. coli has been considered for a long time to only synthesize Mo-bisMGD cofactor, Loschi et al. (81) reported recently the first discovery of a Mo-MPT-dependent enzyme (YedYZ) belonging to the sulfite oxidase family in E. coli. We will thus review the incorporation of Mo-MPT by using the best-studied system, the Rhodobacter capsulatus xanthine dehydrogenase. This enzyme consists of a cytoplasmic heterodimeric complex (α₂β₂) that catalyzes the hydroxylation of hypoxanthine and xanthine, the last two steps in purine degradation (51). The XdhA subunit contains two [2Fe-2S] clusters in addition to flavin adenine dinucleotide (FAD), while the XdhB subunit binds Mo-MPT (72) as confirmed by the crystal structure (152). A distinctive feature compared with other Mo-MPT-dependent enzymes belonging to the sulfite oxidase family is the presence of a sulfur ligand at the Mo atom (152). Functional synthesis of the R. capsulatus XdhAB complex requires the presence of an additional protein, XdhC (33 kDa), encoded by the xdhABC operon (73). Leimkühler and Klipp (73) demonstrated that, contrary to the [Fe-S] clusters and the FAD cofactor, the incorporation of MPT specifically depends on the presence of XdhC. Interestingly, heterologous expression of the R. capsulatus XdhAB subunits in E. coli leads to the synthesis of an inactive enzyme with a full content in Mo-MPT (71). Chemical analysis of the Moco present in the purified heterologous enzymes clearly indicated that XdhC is required for the sulfuration of Moco but not for its insertion, a situation which differs markedly from its absolute requirement for cofactor insertion into XdhAB in the native host R. capsulatus (73). Similarly, Ivanov et al. (55) reported that XdhC is the critical factor for functional heterologous expression of Comamonas acidovorans xanthine dehydrogenase in E. coli. Biochemical studies further indicated that Moco sulfuration is an oxygen-sensitive process (55, 94). In this context, XdhC was shown in vitro to tightly bind MPT in stoichiometric amounts and, as such, protects the sulfurated form of MPT from oxidation (94). In addition, by using a fully defined in vitro system, XdhC was demonstrated to protect and insert sulfurated Moco into apoXdhAB through a direct protein interaction with the catalytic subunit XdhB (94). Altogether, these results are interpreted in terms of a model where XdhC binds Moco and facilitates its sulfuration through the direct interaction with an L-cysteine desulfurase (95). As such, Moco-loaded XdhC specifically interacts with the apoXdhAB complex bearing all its redox centers with the exception of Moco. During this step, where the action of XdhC may be restricted not only to Moco transfer, XdhAB undergoes a conformational change and displays enzymatic activity.

In summary, XdhC differs markedly from the reported function of the above-mentioned enzyme-specific chaperones, which do not play an active role in Moco incorporation but rather allow a multiprotein complex in charge of Moco delivery to interact with their apo-Mo-enzyme partner. Such differences may be explained by the specific requirement of a sulfuration step of Moco, an oxygen-sensitive process, prior its incorporation in the XDH complex. It would be interesting, therefore, to compare the situation with a Mo-MPT enzyme of the sulfite oxidase family that does not require sulfuration of the cofactor. To date, no enzyme-specific chaperones have been reported to be involved in maturation of such enzymes. In particular, E. coli YedY is a soluble periplasmic oxidoreductase which contains Mo-MPT as the unique redox cofactor as disclosed by the crystal structure (81). Accordingly, it is highly probable that biogenesis of such a Tat substrate benefits from the action of a yet unidentified enzyme-specific chaperone in the same manner as the TMAO reductase.

CONCLUDING REMARKS

Our understanding of the biological role and the function of Mo is progressing rapidly. Now that most of the relevant genes are cloned and the principles of Moco biosynthesis are known, research concentrates on the steps beyond Moco biosynthesis, i.e., studying the release of Moco from the biosynthetic complex, its transfer and possible storage, and insertion of Moco into its diverse target enzymes. The latter can be grouped into two classes. Class 1 is represented by monomeric enzymes (Tor, Dor, and Bis) harboring Moco as the sole redox cofactor. Class 2 represents those enzymes (Nar, Fdn, and Dms) that are multimeric and harbor a [Fe-S] cluster in addition to Moco in their catalytic subunit. Genomic analyses indicate that most of prokaryotic Mo-enzymes fall into class 2. Accordingly, their enzyme-specific chaperones are markedly different: chaperones specific for class 1 enzymes only facilitate Moco insertion (with TorD as the prototype), while those specific for class 2 enzymes (with NarJ as the prototype) orchestrate incorporation of both the [Fe-S] cluster and Moco into the catalytic subunit. There are several open questions to address. How is the multienzyme complex for Moco biosynthesis organized? What is the detailed mechanism of Moco insertion into target-enzymes? In particular, how is Moco transferred from the multienzyme complex to the different apoenzymes, and what is the exact function played by class 2 enzyme-specific chaperones in this process? How is Moco sulfurated for enzymes that need a terminal sulfur ligand in the Mo center? How is Moco biosynthesis regulated to meet the changing demands of the cell for Moco? The coming years will bring insight into the integration and (perhaps unexpected) regulatory connections of Moco biosynthesis and Mo-enzymes within the metabolic and physiological network of the cell.

ACKNOWLEDGMENTS

We are grateful to all colleagues who made results available to us prior to publication. We apologize to all those whose important contributions to the field could not be cited because of space limitations.

A.M. acknowledges support from the Centre National de la Recherche Scientifique and the Agence Nationale de la Recherche. R.R.M. acknowledges support from the Deutsche Forschungsgemeinschaft.